Taxol was first isolated in 1971 from the western yew, Taxus brevifolia by Wani, et al. Taxol is a member of the taxane family of diterpenes. Taxanes are diterpene compounds which find utility in the pharmaceutical field. Taxanes may be found in plant materials, and having been isolated therefrom. Paclitaxel is a well known chemotherapeutic drug for treatment of various metastatic cancers. It has been approved for the treatment of ovarian and breast cancers.
Taxal and various taxane derivatives are highly cytotoxic and possess strong in vivo activities in a number of leukemic and tumor systems. Taxol is considered to be an exceptionally promising cancer chemotherapeutic agent.
The only currently available source for taxol is several species of very slow growing yew (genus Taxus, family Taxaceae). Paclitaxel is a natural product, primarily extracted from the bark of the Pacific yew tree, Taxus brevifolia, and is also found in T. baccata, T. walichiana, T. yunnanensis and T canadensis. The isolation procedures currently practised are very difficult and low-yielding. The extraction of taxol from trees of the Taxus genus using liquid methanol has been reported. However, taxanes are generally present in plant materials in relatively small amounts so that, in the case of taxol, for example, large numbers of the slow-growing yew trees forming a source for the compound may be destroyed. Further, large amounts of organic solvents may be employed in a conventional liquid extraction, which may be time consuming as well.
The concentration of paclitaxel in various raw materials is typically low, for example, on the order of between about 0.0004 and about 0.01% (w/w) in the bark of Pacific yew. Such low concentration render the extraction and purification of the compound to pharmaceutical grade from raw materials very challenging, and heretofore impractical on a commercial scale. Various normal phase chromatography techniques have been developed to purify paclitaxel from a crude extract of raw material.
The success of low pressure chromatography greatly depends on the nature of the column. Various problems are associated with the use of silica gel and alumina trioxide, all of which are classical supports of the stationary phase in partition system. They form a stable stationary phase with most solvent systems, but is a strong absorbent and may participate in the separation process to the extent that chromatographic behaviour and recovery of samples are affected.
Chromatography methods have been developed to detect and isolate paclitaxel from various Taxus species on analytical and preparative basis. These isolation processes are mainly conducted on a small laboratory scale and suffer from low selectivity, recovery and high production cost, thereby presenting a serious and unfulfilled need for an economically practicable method for separating the valuable anti-tumor compound paclitaxel from its close analog cephalomanine as well as other closely related taxanes.
Prior art methods disclose the use of various types of chromatographic techniques to separate paclitaxel and related taxanes, including normal phase and reverse phase chromatography on a silica gel or bonded silica gel column. The prior art methods are end up at low yield, high production cost or involved multiple steps which were difficult to scale up to large industrial scale production.
The use of cultivable and renewable plant parts, such as the leaves (needles) and twigs of Taxus species should be the most practical and attractive way of increasing the supply of paclitaxel. The needles of several Taxus species, including Taxus canadensis, have been investigated and found to contain paclitaxel in amounts comparable to the bark of Taxus brevifolia. 
Taxus canadensis is an evergreen shrub found in Eastern Canada and Northeastern United States. This species is unique in its taxane content. The needles contain a major taxane, 9-dihydro-13-acetylbaccatin III (9-DHAB III, 4) along with paclitaxel (0.009–0.05%), 10-deacetylbaccatin III (10-DAB III, 6), baccatin III, (5), cephalomannine, (3), and other minor taxanes. The concentration of (9-DHAB III in the needles is reportedly seven to ten times the concentration of paclitaxel. It appears that (9-DHAB III may be become an important precursor to a new class of semi-synthetic chemotherapeutic agents with increased water solubility.
The production of taxol from ornamental yew needles, barks and roots at present is not economical due to an extremely high percentage of unwanted impurities carried forward in the extract (about 40 to 50% by weight of the dried plant material) during the extraction. This unusually high percentage of impurities in the solvent extract at the needles of ornamental yew makes it very expensive and uneconomical to purify taxol and taxanes from this source in addition to the high cost in drying the needles.
The current procedures are lengthy, costly, or are practically limited to analytical scale. Since paclitaxel occurs in low levels in needles and the needles contains large amounts of waxes, the isolation and purification of paclitaxel from needles to a clinically acceptable purify pose additional challenges. A daunting task is the separation of paclitaxel from its closely related analogue cephalomannine which occurs in the needles and bark. The two analogues have been separated by selective chemical transformation of cephalomannine in a mixture containing both cephalomannine and paclitaxel. Disadvantages associated with these procedures include additional cost from the use of expensive, sometimes toxic, reagents, additional chromatography required to separate the transformed cephalomannine from paclitaxel, the destruction of cephalomannine and sometimes paclitaxel is during the process, and additional chemical transformations which are necessary for recovery of cephalomannine.
Thus, the art has continued to search for ever more efficient and environmentally safe methods for obtaining taxanes which minimize the use of plant materials and organic solvents.
The number of publications and patents describing the isolation and purification of paclitaxel and taxanes from Taxus specie is increasing, but the procedures currently known for isolation paclitaxel are very complex and difficult with a low yield. These patents described various isolation technology, from normal phase chromatography to reverse phase chromatography. The yields of paclitaxel from various species of Taxus genus range from 0.005–0.017%.
U.S. Pat. No. 5,019,504, issued 1991, to Christen, provided a culture process using Taxus brov.folia for producing the taxanes which were then separated by chromatography, by solvent extraction or adsorption methods. Culturing of plant cells is a difficult method for production of the taxanes for use on a large scale.
U.S. Pat. No. 5,380,916, issued Jan. 10, 1995, to Rao, provided a method for isolating certain clinically important taxane derivatives from the crude extract of a naturally occurring Taxus species. The method includes treating the extract by reverse phase liquid chromatography on an adsorbent. The taxane derivatives were caused to be adsorbed on the adsorbent. The taxane derivatives were recovered from the adsorbent by elution with an elutant. The patentee specifically taught the use of silica gel based reverse phase particles, e.g., hydrocarbon-bonded silica gel, cyano bonded silica gel, or phenylalkyl-bonded silica gel. Among the eluents taught were a polar elutant, e.g., lower alcohol, acetone, acetic acid, and acetonitrile, especially a water-miscible organic solvent, e.g., acetonitrile or methanol, in water.
There are many problems with the Rao et al method described hereinabove in U.S. Pat. No. 5,380,916. The most important was that plant lipid components and large quantities of chlorophyll were extracted with about 25% by volume ethanol. These lipid and chlorophyll components interfere with the separation in a chromatographic column. The crude product was colored from compounds in the plant material and these colored compounds interfered with the chromatographic separation. The extraction process of this Rao et al, U.S. Pat. No. 5,380,916, produced large quantities of crude extract in which taxol and taxanes were only a minor component. The reverse phase chromatographic separation using the process of this Rao et al U.S. Pat. No. 5,380,916, was such that taxol was not cleanly separated. The mother liquor from the initial separation of the taxol was subjected to additional reverse phase chromatography and recrystallization to separate more taxol. The more polar solvent fractions contain 10-decetylbaccatin III which can be crystallized to remove this compound and then resubjected to standard chromatography. Multiple reverse phrase liquid chromatographic steps may be necessary in the process of this Rao et al U.S. patent.
Rao et al in U.S. Pat. No. 5,380,916, issued Jan. 10, 1998, also described the use of ligroin to remove lipid components. Aqueous methanol removes the crude taxane mixture, which was then extracted with less polar solvents to separate taxol and related compounds from 10 deacetylbaccatin III. The taxanes can be isolated and recrystallized using the reverse phase chromatography. An aqueous methanol extract was partitioned between water and benzene and then extracted with chloroform and the solvents are removed. Methanol or acetonitrile and water was used in reverse phase liquid chromatography to separate the taxanes. However, none of the procedures taught by Rao in U.S. Pat. No. 5,380,916 provided a clean separation of each of the components in a single pass through a column without a solvent-solvent extraction. Large amounts of various solvents are necessary.
U.S. Pat. No. 5,407,674, issued Apr. 18, 1995, to Gabetta, et al., provided a procedure for the isolation of a taxane derivative. The procedure included first extracting the vegetal material of yew cultivar with at least one chlorinated hydrocarbon or mixtures of at least one chlorinated hydrocarbon, e.g., methylene chloride, and at least one lower alkanol, e.g., methanol. In a second step chromatographing the residue obtained in the first step with an eluent comprising a mixture of at least one aliphatic hydrocarbon or aromatic hydrocarbon and at least one solvent having a higher polarity than the hydrocarbon. For example, when the residue is chromatographed on silica gel as a stationary phase and the eluent comprises toluene as the aromatic hydrocarbon. Acetone or diisopropyl ether etherone in admixture, with n-hexane or n-heptane is the solvent having a higher polarity.
U.S. Pat. No. 5,620,875, issued Apr. 15, 1997, to Hoffman et al. taught the separation of paclitaxel and other taxanes by multiple step hexane extractions and high performance liquid chromatography (HPLC). The method is involved, labor intensive and only provides moderate yields of the desired compounds.
U.S. Pat. No. 5,670,673, issued Sep. 23, 1997, to Rao, taught the isolation and purification of taxol and its analogues. The method included the use of reverse phase liquid chromatography on a C18 adsorbent with elution of the adsorbed analogues. Limitations exist with this in terms of productivity and purity of compounds obtained. Reverse phase chromatographic separation of impure taxanes from plant materials is very expensive because of the cost of the column materials. Generally, reverse phase separation can be used on the bark of Pacific Yew because of the relatively low concentration of pigments, lipids and waxes and high concentration of taxol. However, the yew needles contain lesser amounts of taxol and significant amounts of impurities and thus reverse phase chromatography for separation of taxol from the bulk of the yew materials other than Pacific yew bark is not practical. The production of taxol by such prior art technique is encumbered by time consuming extraction and purification procedures; long residence times in a harsh environment; and low overall yields.
U.S. Pat. No. 6,503,396, issued Jan. 7, 2003, to Kim, et al. provided methods for isolating taxol and derivatives thereof with high purity. The method for isolating taxol or derivatives thereof from a source material included the first step of extracting the source material with a supercritical fluid, e.g., carbon dioxide and a cosolvent to obtain an extract, e.g., a mixture of water and methanal or ethanol. It included the second step of liquid-liquid separating the extract with an organic solvent, e.g., (n-hexane, to obtain a solvent layer. It included the final step of isolating taxol or the derivatives thereof from the solvent layer by column chromatography, e.g., using multiple columns to isolate taxol or derivatives, wherein each of said multiple columns comprises a column resin, e.g., silica gel, RP-18, or Sephadex.
Canadian Patent No. 2,126,698, issued Nov. 8, 1993, in the name of Nair, provided a method for the separation of taxanes, particularly taxol, cephalomannine, baccatin III and deacetylbaccatin III, from ornamental yew tissue. The method involved the use of a specific solvent mixture of water and about 50 to 95 percent by volume methanol, ethanol or acetone, and the treatment of the resulting extract using activated carbon, preferably charcoal. The taxanes were preferably separated from the crude extract by a normal phase chromatographic step which preferably was through vacuum and then medium pressure column chromatographic separation, using inexpensive silica gel as an absorbent. The silica gel was regenerated by heating in a furnace above above 500° C. to remove adsorbed organic materials.
Canadian Patent No. 2,157,905, issued Mar. 18, 1994, in the name of Durand et al, provided a method for purifying a method for purifying taxcoids by centrifugal partition chromatography using a mixture of aliphatic hydrocarbons, esters, or alcohols and water having a particular partition coefficient in one phase and having a different partition coefficient in a second phase.
Canadian Patent 2,213,952, issued Jun. 15, 1999, in the name of G. Caron, taught the isolation of paclitaxel and (9-dihydro-1-13-acetylbaccatin III from Taxus canadensis by a combination of solvent precipitation and multiple steps of reverse phase HPLC. The methods used in that patent are too complicated and very difficult for industrial scale production. It is also assumed various and large quantities of organic solvent, some of those solvents are toxic and expensive. It is raised environmental concerns about the emission of the organic solvents' gaseous effluent. Also using ODS-2 as a preparative column packing material for industrial production is expensive and increases the production cost. To make a silylated paclitaxel derivative to reach final purity is not feasible in industrial scale and involves extra cost for these purification steps.